The present invention relates to polynucleotides which encode plant polypeptides and which exhibit senescence-induced expression, transgenic plants containing the polynucleotides in antisense orientation and methods for controlling senescence in plants. More particularly, the present invention relates to plant lipase genes whose expression is induced by the onset of senescence and the use of the lipase gene to control senescence in plants.
Senescence is the terminal phase of biological development in the life of a plant. It presages death and occurs at various levels of biological organization including the whole plant, organs, flowers and fruit, tissues and individual cells.
Cell membrane deterioration is an early and fundamental feature of senescence. Metabolism of lipids, in particular membrane lipids, is one of several biochemical manifestations of cellular senescence. Rose petals, for example, sustain an increase in acyl hydrolase activity as senescence progresses that is accompanied by a loss of membrane function (Borochov, et al., Plant Physiol., 1982, 69, 296-299). Cell membrane deterioration is an early and characteristic feature of senescence engendering increased permeability, loss of ionic gradients and decreased function of key membrane proteins such as ion pumps (Brown, et al., Plant Physiol.: A Treatise, Vol. X. Academic Press, 1991, pp.227-275). Much of this decline in membrane structural and functional integrity can be attributed to lipase-mediated phospholipid metabolism. Loss of lipid phosphate has been demonstrated for senescing flower petals, leaves, cotyledons and ripening fruit (Thompson, J. E., Senescence and Aging in Plants, Academic Press, San Diego, 1988, pp. 51-83), and this appears to give rise to major alterations in the molecular organization of the membrane bilayer with advancing senescence that lead to impairment of cell function. In particular, studies with a number of senescing plant tissues have provided evidence for lipid phase separations in membranes that appear to be attributable to an accumulation of lipid metabolites in the membrane bilayer (McKersie and Thompson, 1979, Biochim. Biophys. Acta, 508: 197-212; Chia, et al., 1981, Plant Physiol., 67:415-420). There is growing evidence that much of the metabolism of lipids in senescing tissue is achieved through senescence-specific changes in gene expression (Buchanan-Wollaston, V., J. Exp. Bot., 1997, 307:181-199).
The onset of senescence can be induced by different factors both internal and external. For example, ethylene plays a role in many plants in a variety of plant processes such as seed germination, seedling development, fruit ripening and flower senescence. Ethylene production in plants can also be associated with trauma induced by mechanical wounding, chemicals, stress (such as produced by temperature and water amount variations), and by disease. Ethylene has been implicated in the regulation of leaf senescence in many plants, but evidence obtained with transgenic plants and ethylene response mutants has indicated that, although ethylene has an effect on senescence, it is not an essential regulator of the process. In many plants ethylene seems to have no role in fruit ripening or senescence. For example in the ripening of fruits of non-climacteric plants such as strawberry, in senescence of some flowers such as day lilies and in leaf senescence in some plants, such as Arabidopsis, and in particular, in the monocots there is no requirement for ethylene signaling (Smart, C. M., 1994, New Phytology, 126:419-448; Valpuesta, et al., 1995, Plant Mol. Biol., 28:575-582).
External factors that induce premature initiation of senescence include environmental stresses such as temperature, drought, poor light or nutrient supply, as well as pathogen attack. As in the case of natural (age-related) senescence, environmental stress-induced senescence is characterized by a loss of cellular membrane integrity. Specifically, exposure to environmental stress induces electrolyte leakage reflecting membrane damage (Sharom, et al., 1994, Plant Physiol., 105:305-308; Wright and Simon, 1973, J. Exp. Botany, 24:400-411; Wright, M., 1974, Planta,120:63-69; and Eze et al., 1986, Physiologia Plantarum, 68:323-328), a decline in membrane phospholipid levels (Wright, M., 1974, Planta,120:63-69) and lipid phase transitions (Sharom, et al., 1994, Plant Physiol., 105:305-308), all of which can be attributed to the action of lipase. Plant tissues exposed to environmental stress also produce ethylene, commonly known as stress ethylene (Buchanan-Wollaston, V., 1997, J. Exp. Botany, 48.181-199; Wright, M., 1974, Planta,120:63-69). As noted above, ethylene is known to cause senescence in some plants. Membrane deterioration leading to leakage is also a seminal feature of seed aging, and there is evidence that this too reflects deesterification of fatty acids from membrane phospholipids (McKersie, B. D., Senarata, T., Walker, M. A., Kendall, E. J. and Hetherington, P. R. In: Senescence and Aging in Plants, Ed. L. D. Nooden and A. C. Leoopold, academic Press, 1988. PP 441-464).
Presently, there is no widely applicable method for controlling onset of senescence caused by either internal or external, e.g., environmental stress, factors. At present, the technology for controlling senescence and increasing the shelf-life of fresh, perishable plant produce, such as fruits, flowers and vegetables relies primarily upon reducing ethylene biosynthesis. For example, U.S. Pat. No. 5,824,875 discloses transgenic geranium plants which exhibit prolonged shelf-life due to reduction in levels of ethylene resulting from the expression of one of three 1-amino-cyclopropane-1-carboxylate (ACC) synthase genes in antisense orientation. Consequently, this technology is applicable to only a limited range of plants that are ethylene-sensitive.
The shelf-life of some fruits is also extended by reducing ethylene biosynthesis, which causes ripening to occur more slowly. Since senescence of these fruits is induced after ripening, the effect of reduced ethylene biosynthesis on shelf-life is indirect. Another approach used to delay fruit ripening is by altering cellular levels of polygalacturonase, a cell-wall softening enzyme that is synthesized during the early stages of ripening. This approach is similar to controlling ethylene biosynthesis in that it, too, only indirectly affects senescence and again, is only applicable to a narrow range of plants.
Thus, there is a need for a method of controlling senescence in plants which is applicable to a wide variety of plants. It is therefore of interest to develop senescence modulating, technologies that are applicable to all types of plants, regardless of ethylene sensitivity.
This invention is based on the discovery and cloning of a full length cDNA clone encoding a carnation senescence-induced lipase and a full-length cDNA clone encoding Arabidopsis thaliana senescence-induced lipase. The nucleotide sequences and corresponding amino acid sequences for the senescence-induced lipase genes are disclosed herein. The nucleotide sequence of the carnation senescence-induced lipase gene has been successfully used as a heterologous probe to detect corresponding genes or RNA transcripts in several plants that are similarly regulated.
The invention provides a method for genetic modification of plants to control the onset of senescence, either age-related senescence or environmental stress-induced senescence. The senescence-induced lipase nucleotide sequences of the invention, fragments thereof, or combinations of such fragments, are introduced into a plant cell in reverse orientation to inhibit expression of the endogenous senescence-induced lipase gene, thereby reducing the level of endogenous senescence-induced lipase and altering senescence in the transformed plant.
Using the methods of the invention, transgenic plants are generated and monitored for growth and development. Plants or detached parts of plants (e.g., cuttings, flowers, vegetables, fruits, seeds or leaves) exhibiting prolonged life or shelf life with respect to plant growth, flowering, reduced fruit spoilage, reduced seed aging and/or reduced yellowing of leaves due to reduction in the level of senescence-induced lipase are selected as desired products having improved properties including reduced leaf yellowing, reduced petal abscission, reduced fruit spoilage during shipping and storage. These superior plants are propagated. Similarly, plants exhibiting increased resistance to environmental stress, e.g., decreased susceptibility to low temperature (chilling), drought, infection, etc., are selected as superior products.
In one aspect, the present invention is directed to an isolated DNA molecule encoding senescence-induced lipase, wherein the DNA molecule hybridizes with SEQ ID NO:1, or a functional derivative of the isolated DNA molecule which hybridizes with SEQ ID NO:1. In one embodiment of the invention, the isolated DNA molecule has the nucleotide sequence of SEQ ID NO:1, i.e., 100% complementarity (sequence identity) to SEQ ID NO:1. In another embodiment of this aspect of the invention, the isolated DNA molecule contains the nucleotide sequence of SEQ ID NO:4.
The invention is also directed to an isolated DNA molecule encoding senescence-induced lipase, wherein the DNA molecule hybridizes with SEQ ID NO:18, or a functional derivative of the isolated DNA molecule which hybridizes with SEQ ID NO:18. In one embodiment of this aspect of the invention, the isolated DNA molecule has the nucleotide sequence of SEQ ID NO:18, i.e., 100% complementarity (sequence identity) to SEQ ID NO:18. In another embodiment of this aspect of the invention, the isolated DNA molecule contains the nucleotide sequence of SEQ ID NO:19.
In another embodiment of the invention, there is provided an isolated protein encoded by a DNA molecule as described herein above, or a functional derivative thereof. A preferred protein has the amino acid sequence of SEQ ID NO:2, or is a functional derivative thereof.
Also provided herein is an antisense oligonucleotide or polynucleotide encoding an RNA molecule which is complementary to at least a portion of an RNA transcript of the DNA molecule described hereinabove, wherein the RNA molecule hybridizes with the RNA transcript such that expression of endogenous senescence-induced lipase is altered. The antisense oligonucleotide or polynucleotide can be full length or preferably has about six to about 100 nucleotides.
The antisense oligonucleotide or polynucleotide is substantially complementary to a corresponding portion of one strand of a DNA molecule encoding senescence-induced lipase, wherein the DNA molecule encoding senescence-induced lipase hybridizes with SEQ ID NO:1, SEQ ID NO:18 or both, or is substantially complementary to a corresponding portion of an RNA sequence encoded by the DNA molecule encoding senescence-induced lipase. In one embodiment of the invention, the antisense oligonucleotide or polynucleotide is substantially complementary to a corresponding portion of one strand of the nucleotide sequence SEQ ID NO:1,SEQ ID NO:18 or both or the RNA transcript encoded by SEQ ID NO:1. In another embodiment, the antisense oligonucleotide is substantially complementary to a corresponding portion of about 100 to about 200 nucleotides of the 5xe2x80x2 non-coding portion or 3xe2x80x2-end portion of one strand of a DNA molecule encoding senescence-induced lipase, wherein the DNA molecule hybridizes with SEQ ID NO:1, SEQ ID NO:18 or both. In another embodiment, the antisense oligo- or polynucleotide is substantially complementary to a corresponding portion of the open reading frame of one strand of the nucleotide sequence SEQ ID NO:4 or the RNA transcript encoded by SEQ ID NO:4.
The invention is further directed to a vector for transformation of plant cells, comprising
(a) antisense nucleotide sequences substantially complementary to (1) a corresponding portion of one strand of a DNA molecule encoding senescence-induced lipase, wherein the DNA molecule encoding senescence-induced lipase hybridizes with SEQ ID NO:1,SEQ ID NO:18 or both or (2) a corresponding portion of an RNA sequence encoded by the DNA molecule encoding senescence-induced lipase; and
(b) regulatory sequences operatively linked to the antisense nucleotide sequences such that the antisense nucleotide sequences are expressed in a plant cell into which it is transformed.
The regulatory sequences include a promoter functional in he transformed plant cell, which promoter may be inducible or onstitutive. Optionally, the regulatory sequences include a olyadenylation signal.
The invention also provides a plant cell transformed with the vector as described above, a plantlet or mature plant generated from such a cell, or a plant part of such a plantlet or plant.
The present method is further directed to a method of producing a plant having a reduced level of senescence-induced lipase compared to an unmodified plant, comprising:
(1) transforming a plant with a vector as described above;
(2) allowing the plant to grow to at least a plantlet stage;
(3) assaying the transformed plant or plantlet for altered senescence-induced lipase activity and/or altered senescence and/or altered environmental stress-induced senescence and/or ethylene-induced senescence; and
(4) selecting and growing a plant having altered senescence-induced lipase activity and/or altered senescence and/or altered environmental stressed-induced senescence or ethylene-induced senescence compared to an nom-transformed plant.
A plant produced as above, or progeny, hybrids, clones or plant parts preferably exhibit reduced senescence-induced lipase expression and delayed senescence and/or delayed stress-induced senescence or ethylene-induced senescence.
This invention is further directed to a method of inhibiting expression of endogenous senescence-induced lipase in a plant cell, said method comprising:
(1) integrating into the genome of a plant a vector comprising
A) antisense nucleotide sequences complementary to (i) a corresponding portion of one strand of a DNA molecule encoding endogenous senescence-induced lipase, wherein the DNA molecule encoding the endogenous senescence-induced lipase hybridizes with SEQ ID NO:1, SEQ ID NO:18 or both, or (ii) a corresponding portion of an RNA sequence encoded by the endogenous senescence-induced lipase gene; and
(B) regulatory sequences operatively linked to the antisense nucleotide sequences such that the antisense nucleotide sequences are expressed; and
(2) growing said plant, whereby said antisense nucleotide sequences are transcribed and the transcript binds to said endogenous RNA whereby expression of said senescence-induced lipase gene is inhibited.